Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb 22;11(2):e0149676.
doi: 10.1371/journal.pone.0149676. eCollection 2016.

The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes Through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures

Affiliations
Free PMC article

The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes Through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures

Yuri Sakamoto et al. PLoS One. .
Free PMC article

Abstract

Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance, and peroxisome proliferator-activated receptors (PPARs) regulate inflammatory gene expression in these cells. Recently, the soy isoflavone daidzein was reported to act as a PPAR activator. We examined whether daidzein affected adipocyte-macrophage crosstalk via the regulation of PPARs. Co-cultures of 3T3-L1 adipocytes and RAW264 macrophages, or palmitate-stimulated RAW264 macrophages were treated with daidzein in the presence or absence of specific inhibitors for PPARs: GW6471 (a PPARα antagonist), and GW9662 (a PPARγ antagonist). Inflammatory gene expression was then determined. Daidzein significantly decreased chemokine (C-C motif) ligand 2 (Ccl2, known in humans as monocyte chemo-attractant protein 1 (MCP1)) and interleukin 6 (Il6) mRNA levels induced by co-culture. In 3T3-L1 adipocytes, daidzein inversed the attenuation of adiponectin gene expression by co-culture, and these effects were inhibited by the PPAR-γ specific inhibitor. Daidzein also decreased Ccl2 and Il6 mRNA levels in RAW264 macrophages stimulated with palmitate or conditioned medium (CM) from hypertrophied 3T3-L1 adipocytes. This inhibitory effect on Il6 expression was abrogated by a PPAR-α inhibitor. Additionally, we examined the activation of nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways and found that daidzein significantly inhibited palmitate-induced phosphorylation of JNK. Our data suggest that daidzein regulates pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the JNK pathway in adipocyte and macrophage co-cultures. These effects might be favorable in improving adipose inflammation, thus, treatment of daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of daidzein on pro-inflammatory gene expression in contact co-culture of 3T3-L1 adipocytes and RAW264 macrophages.
3T3-L1 preadipocytes were differentiated for seven days. RAW264 macrophages were co-cultured on adipocytes (Co-culture) in serum-free medium with (Daid) or without daidzein (Cont) for 24 h. (A) Ccl2, Il6, Tnf and adiponectin mRNA levels were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with a separate Cont culture arbitrarily set to 1. (B) The amounts of CCL-2, and IL-6 protein in the medium were determined by ELISA and normalized to the total cell protein content. **, p<0.01 versus Cont; §, p<0.05; §§, p<0.01 versus co-culture Cont.
Fig 2
Fig 2. Effect of daidzein via PPAR-γ inhibition on pro-inflammatory gene expression in transwell co-cultured adipocytes.
3T3-L1 preadipocytes were differentiated for seven days. RAW264 macrophages were plated onto the transwell insert for 24 h. 3T3-L1 cells were differentiated in DMSO (Cont), 25 μM of daidzein (Daid), or Daid + 10 μM of GW9662 (Daid+GW). Ccl2, Il6 and adiponectin mRNA levels in 3T3-L1 adipocytes were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with a Cont culture without macrophages arbitrarily set to 1. **, p<0.01 versus Cont; Δ, p <0.1 versus co-culture Cont; #, p<0.05 versus co-culture Daid.
Fig 3
Fig 3. Effect of daidzein on PPAR-α and PPAR-γ activity in HEK293T cells.
HEK293T cells were transfected with PPRE-containing reporter plasmid and (A) PPAR-α or (B) PPAR-γ expression plasmids, and then treated with the indicated concentrations (6.25–25 μM) of daidzein for 24 h. Luciferase activity was measured and expressed as a fold induction, that was corrected for transfection efficiency using renilla luciferase activity. Values are expressed as the fold change compared with the vehicle control that was arbitrarily set to 1. Δ, p<0.1; *, p<0.05; **, p<0.01 versus non-treated control.
Fig 4
Fig 4. Effects of daidzein via PPAR-α/γ inhibition in palmitate- and CM- treated macrophages.
(A) RAW264 macrophages were cultured in the serum-free medium with the indicated concentrations (6.25–25 μM) of daidzein for 1 h, then stimulated with 400 μM palmitate for 4 h. (B) Cells were cultured in serum-free medium with DMSO (Cont), 25 μM of daidzein (Daid), Daid + 10 μM of GW6471 (Daid+α) or Daid + 10 μM of GW9662 (Daid+γ) for 1 h, then 400 μM palmitate was added and cells were incubated for another 4 h. (C) Cells were cultured in CM derived from hypertrophied adipocytes with Daid, Daid+α or Daid+γ for 24 h. Messenger RNA levels of each gene were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with the vehicle control that was arbitrarily set to 1. *, p<0.05; **, p<0.01 versus Cont. §, p<0.05; §§, p<0.01 versus palmitate Cont or CM Cont. Δ, p<0.1; ##, p<0.01 versus palmitate Daid or CM Daid.
Fig 5
Fig 5. Effect of daidzein on palmitate-induced phosphorylation of JNK and p65 in RAW264 macrophages.
RAW264 macrophages were cultured in serum-free medium with (Daid) or without daidzein (Cont) for 1 h. Palmitate was added and cells were incubated for another 4 h. (A) Cells lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from two separate experiments with similar results is shown. (B) Densitometric analysis of protein bands in the western blots was done using Image J software. Optical density for each phosphorylated protein was calculated and normalized with the value of each total protein. **, p<0.01 versus Cont. §§, p<0.01 versus palmitate Cont.
Fig 6
Fig 6. Putative mechanisms for the anti-inflammatory effect of daidzein on adipocyte-macrophage co-cultures.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

References

    1. Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest. 2005;115(5):1111–1119. - PMC - PubMed
    1. Hotamisligil GS. Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord. 2003;27 Suppl 3:S53–55. - PubMed
    1. Wellen KE, Hotamisligil GS. Obesity-induced inflammatory changes in adipose tissue. J Clin Invest. 2003;112(12):1785–1788. - PMC - PubMed
    1. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112(12):1796–1808. - PMC - PubMed
    1. Trayhurn P, Wood IS. Adipokines: inflammation and the pleiotropic role of white adipose tissue. Br J Nutr. 2004;92(3):347–355. - PubMed

Publication types

Grant support

This study was supported in part by grants-in-aid for Scientific Research (grant nos. 24300254, 26750037, 15J11645 and 15H02895) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Feedback